A longitudinal, multi-parametric functional MRI study to determine age-related changes in the rodent brain.

As the population ages, the incidence of age-related neurological diseases and cognitive decline increases. To further understand disease-related changes in brain function it is advantageous to examine brain activity changes in healthy aging rodent models to permit mechanistic investigation. Here, we examine the suitability, in rodents, of using a novel, minimally invasive anaesthesia protocol in combination with a functional MRI protocol to assess alterations in neuronal activity due to physiological aging. 11 Wistar Han female rats were studied at 7, 9, 12, 15 and 18 months of age. Under an intravenous infusion of propofol, animals underwent functional magnetic resonance imaging (fMRI) and functional magnetic resonance spectroscopy (fMRS) with forepaw stimulation to quantify neurotransmitter activity, and resting cerebral blood flow (CBF) quantification using arterial spin labelling (ASL) to study changes in neurovascular coupling over time. Animals showed a significant decrease in size of the active region with age (P â€‹< â€‹0.05). fMRS results showed a significant decrease in glutamate change with stimulation (ΔGlu) with age (P â€‹< â€‹0.05), and ΔGlu became negative from 12 months onwards. Global CBF remained constant for the duration of the study. This study shows age related changes in the blood oxygen level dependent (BOLD) response in rodents that correlate with those seen in humans. The results also suggest that a reduction in synaptic glutamate turnover with age may underlie the reduction in the BOLD response, while CBF is preserved.

Perioperative Cerebral Microbleeds After Adult Cardiac Surgery.

Background and Purpose- Cerebral microbleeds (CMBs) have been observed using magnetic resonance imaging in patients with cardiovascular risk factors, cognitive deterioration, small vessel disease, and dementia. They are a well-known consequence of cerebral amyloid angiopathy, chronic hypertension, and diffuse axonal injury, among other causes. However, the frequency and location of new CMBs postadult cardiac surgery, in association with cognition and perioperative risk factors, have yet to be studied. Methods- Pre- and postsurgery magnetic resonance susceptibility-weighted images and neuropsychological tests were analyzed from a total of 75 patients undergoing cardiac surgery (70 men; mean age, 63±10 years). CMBs were identified by a neuroradiologist blinded to clinical details who independently assessed the presence and location of CMBs using standardized criteria. Results- New CMBs were identified in 76% of patients after cardiac surgery. The majority of new CMBs were located in the frontal lobe (46%) followed by the parietal lobe (15%), cerebellum (13%), occipital lobe (12%), and temporal lobe (8%). Patients with new CMBs typically began with a higher prevalence of preexisting CMBs ( P=0.02). New CMBs were associated with longer cardiopulmonary bypass times ( P=0.003), and there was a borderline association with lower percentage hematocrit ( P=0.04). Logistic regression analysis suggested a ≈2% increase in the odds of acquiring new CMBs during cardiac surgery for every minute of bypass time (odds ratio, 1.02; 95% CI, 1.00-1.05; P=0.04). Postoperative neuropsychological decline was observed in 44% of patients and seemed to be unrelated to new CMBs. Conclusions- New CMBs identified using susceptibility-weighted images were found in 76% of patients who underwent cardiac surgery. CMBs were globally distributed with the highest numbers in the frontal and parietal lobes. Our regression analysis indicated that length of cardiopulmonary bypass time and lowered hematocrit may be significant predictors for new CMBs after cardiac surgery. Clinical Trial Registration- URL: http://www.isrctn.com . Unique identifier: 66022965.

Impact of perioperative infarcts after cardiac surgery.

BACKGROUND AND PURPOSE: Brain injury after cardiac surgery is a serious concern for patients and their families. The purpose of this study was to use 3-T fluid attenuated inversion recovery MRI to characterize new and preexisting cerebral ischemic lesions in patients undergoing cardiac surgery and to test whether the accumulation of new ischemic lesions adversely affects cognition. METHODS: Digital comparison of before and after fluid attenuated inversion recovery MRI images was performed for 77 cardiac surgery patients. The burden of preexisting versus new ischemic lesions was quantified and compared with the results of baseline and postoperative neuropsychological testing. RESULTS: After surgery, new lesions were identified in 31% of patients, averaging 0.5 lesions per patient (67 mm(3) [0.004%] of brain tissue). Patients with preexisting lesions were 10× more likely to receive new lesions after surgery than patients without preexisting lesions. Preexisting ischemic lesions were observed in 64% of patients, averaging 19.4 lesions (1542 mm(3) [0.1%] of brain tissue). New lesions in the left hemisphere were significantly smaller and more numerous (29 lesions; median volume, 44 mm(3); volume range, 5-404 mm(3)) than those on the right (10 lesions; median volume, 128 mm(3); volume range, 13-1383 mm(3)), which is consistent with a cardioembolic source of particulate emboli. Overall, the incidence of postoperative cognitive decline was 46% and was independent of whether new lesions were present. CONCLUSIONS: New lesions after cardiac surgery added a small (≈4%) contribution to the burden of preexisting cerebrovascular disease and did not seem to affect cognitive function. CLINICAL TRIAL REGISTRATION URL: http://public.ukcrn.org.uk. Unique identifier: UKCRN ID: 11702.

Are portable ankle brachial pressure index measurement devices suitable for hypertension screening?

OBJECTIVE: In a large-scale population cardiovascular screening programme, peripheral artery disease (PAD) and hypertension would ideally be rapidly assessed using a single device. The ankle-brachial pressure index (ABPI) is calculated by comparing the ankle and brachial blood pressure (BP). However, it is currently unclear whether brachial BP measurements provided by automated PAD screening systems are sufficiently accurate for simultaneous hypertension screening. METHODS: Two portable PAD screening devices, the MESI ABPI MD and Huntleigh's Dopplex ABIlity, were evaluated following the European Society of Hypertension International Protocol (ESH-IP) Revision 2010 using a mercury-free sphygmomanometer as a reference device. RESULTS: On average, the MESI slightly underestimated brachial systolic blood pressure (BP) with a bias and standard deviation (SD) of -3.5 (SD: 3.3) mmHg and diastolic BP with a bias of -1.5 (SD: 2.3) mmHg. For systolic BP estimates, the Dopplex was more accurate than the MESI with a lower bias of -0.5 (SD: 4.2) mmHg but less precise. The MESI successfully fulfilled all the requirements of the ESH-IP for hypertension screening. The Dopplex device failed the ESH-IP due to the absence of DBP measurements. CONCLUSIONS: The MESI device appears to be suitable for simultaneous PAD and hypertension screening as part of a preventative care programme. Huntleigh's Dopplex ABIlity failed to pass the ESH-IP validation test. Further clinical trials are underway to assess the use of the MESI for simultaneous screening for hypertension and PAD in a population screening setting.

Development of a Flow Phantom for Transcranial Doppler Ultrasound Quality Assurance.

Anecdotal evidence was recently brought to our attention suggesting a potential difference in velocity estimates between transcranial Doppler (TCD) systems when measuring high velocities (∼200 cm/s) close to the threshold for sickle cell disease stroke prevention. As we were unable to identify a suitable commercial TCD phantom, a middle cerebral artery (MCA) flow phantom was developed to evaluate velocity estimates from different devices under controlled conditions. Time-averaged velocity estimates were obtained using two TCD devices: a Spencer Technologies ST3 Doppler system (ST3 PMD150, Spencer Technologies, Seattle, WA, USA) and a DWL Dopplerbox (DWL Compumedics, SN-300947, Singen, Germany). These were compared with velocity estimates obtained using a Zonare duplex scanner (Zonare Medical Systems, Mountain View, CA, USA), with timed collection of fluid as the gold standard. Bland-Altman analysis was performed to compare measurements between devices. Our tests confirmed that velocities measured with the DWL TCD system were +4.1 cm/s (+3.7%; limits of agreement [LoA]: 2%, 5%; p = 0.03) higher than the Spencer system when measuring a velocity 110 cm/s and +12 cm/s higher (+5.7 %; LoA: 4.8%, 6.6%; p = 0.03) when measuring velocities of 210 cm/s, close to the diagnostic threshold for stroke intervention. We found our MCA phantom to be a valuable tool for systematically quantifying differences in TCD velocity estimates between devices, confirming that the DWL system gave consistently higher readings than the Spencer ST3 system. Differences become more pronounced at high velocities, which explains why they were not identified earlier. Our findings have clinical implications for centers using TCD to monitor patients with sickle cell disease, as extra care may be needed to adjust for bias between manufacturers when making treatment decisions about children with sickle cell with velocities close to the diagnostic threshold.

Mutant p53 promotes RCP-dependent chemoresistance coinciding with increased delivery of P-glycoprotein to the plasma membrane.

TP53 is the most frequently mutated gene in cancers. Mutations lead to loss of p53 expression or expression of a mutant protein. Mutant p53 proteins commonly lose wild-type function, but can also acquire novel functions in promoting metastasis and chemoresistance. Previously, we uncovered a role for Rab-coupling protein (RCP) in mutant p53-dependent invasion. RCP promotes endosomal recycling and signalling of integrins and receptor tyrosine kinases. In a screen to identify novel RCP-interacting proteins, we discovered P-glycoprotein (P-gp). Thus, we hypothesised that mutant p53 could promote chemoresistance through RCP-dependent recycling of P-gp. The interaction between RCP and P-gp was verified endogenously and loss of RCP or mutant p53 rendered cells more sensitive to cisplatin and etoposide. In mutant p53 cells we detected an RCP-dependent delivery of P-gp to the plasma membrane upon drug treatment and decreased retention of P-gp substrates. A co-localisation of P-gp and RCP was seen in mutant p53 cells, but not in p53-null cells upon chemotherapeutic exposure. In conclusion, mutant p53 expression enhanced co-localisation of P-gp and RCP to allow for rapid delivery of P-gp to the plasma membrane and increased resistance to chemotherapeutics.

Longitudinal Multimodal fMRI to Investigate Neurovascular Changes in Spontaneously Hypertensive Rats.

Hypertension is an important risk factor for age-related cognitive decline and neuronal pathologies. Studies have shown a correlation between hypertension, disruption in neurovascular coupling and cerebral autoregulation, and cognitive decline. However, the mechanisms behind this are unclear. To further understand this, it is advantageous to study neurovascular coupling as hypertension progresses in a rodent model. Here, we use a longitudinal functional MRI (fMRI) protocol to assess the impact of hypertension on neurovascular coupling in spontaneously hypertensive rats (SHRs). Eight female SHRs were studied at 2, 4, and 6 months of age, as hypertension progressed. Under an IV infusion of propofol, animals underwent fMRI, functional MR spectroscopy, and cerebral blood flow (CBF) quantification to study changes in neurovascular coupling over time. Blood pressure significantly increased at 4 and 6 months (P < .0001). CBF significantly increased at 4 months old (P < .05), in the acute stage of hypertension. The size of the active region decreased significantly at 6 months old (P < .05). Change in glutamate signal during activation, and N-acetyl-aspartate (NAA) signal, remained constant. This study shows that, while cerebral autoregulation is impaired in acute hypertension, the blood oxygenation-level-dependent (BOLD) response remains unaltered until later stages. At this stage, the consistent NAA and glutamate signals show that neuronal death has not occurred, and that neuronal activity is not affected at this stage. This suggests that neuronal activity and viability is not lost until much later, and changes observed here in BOLD activity are due to vascular effects.

Neurological impact of emboli during adult cardiac surgery.

OBJECTIVES: This study draws on advances in Doppler ultrasound bubble sizing to investigate whether high volumes of macro-bubbles entering the brain during cardiac surgery increase the risk of new cerebral microbleeds (CMBs), ischemic MR lesions, or post-operative cognitive decline (POCD). METHODS: Transcranial Doppler (TCD) ultrasound recordings were analysed to estimate numbers of emboli and macrobubbles (>100 µm) entering the brain during cardiac surgery. Logistic regression was used to explore the hypothesis that emboli characteristics affect the incidence of new brain injuries identified through pre- and post-operative MRI and neuropsychological testing. RESULTS: TCD, MRI, and neuropsychological test data were compared between 28 valve and 18 CABG patients. Although valve patients received over twice as many emboli per procedure [median: 1995 vs. 859, p = .004], and seven times as many macro-bubbles [median: 218 vs. 28, p = .001], high volumes of macrobubbles were not found to be significantly associated with new CMBs, new ischaemic lesions, or POCD. The odds of acquiring new CMBs increased by approximately 5% [95% CI: 1 to 10%] for every embolus detected in the first minute after the release of the aortic cross-clamp (AxC). Logistic regression models also confirmed previous findings that cardiopulmonary bypass time and valve surgery were significant predictors for new CMBs (both p = .03). Logistic regression analysis estimated an increase in the odds of acquiring new CMBs of 6% [95% CI: 1 to 12%] for every minute of bypass time over 91 mins. CONCLUSIONS: This small study provides new information about the properties and numbers of bubbles entering the brain during surgery, but found no evidence to substantiate a direct link between large numbers of macrobubbles and adverse cognitive or MR outcome. Clinical Trial Registration URL - http://www.isrctn.com. Unique identifier: 66022965.

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

A preclinical ultrasound method for the assessment of vascular disease progression in murine models.

INTRODUCTION: The efficacy of preclinical ultrasound at providing a quantitative assessment of mouse models of vascular disease is relatively unknown. In this study, preclinical ultrasound was used in combination with a semi-automatic image processing method to track arterial distension alterations in mouse models of abdominal aortic aneurysm and atherosclerosis. METHODS: Longitudinal B-mode ultrasound images of the abdominal aorta were acquired using a preclinical ultrasound scanner. Arterial distension was assessed using a semi-automatic image processing algorithm to track vessel wall motion over the cardiac cycle. A standard, manual analysis method was applied for comparison. RESULTS: Mean arterial distension was significantly lower in abdominal aortic aneurysm mice between day 0 and day 7 post-onset of disease (p < 0.01) and between day 0 and day 14 (p < 0.001), while no difference was observed in sham control mice. Manual analysis detected a significant decrease (p < 0.05) between day 0 and day 14 only. Atherosclerotic mice showed alterations in arterial distension relating to genetic modification and diet. Arterial distension was significantly lower (p < 0.05) in Ldlr-/- (++/--) mice fed high-fat western diet when compared with both wild type (++/++) mice and Ldlr-/- (++/--) mice fed chow diet. The manual method did not detect a significant difference between these groups. CONCLUSIONS: Arterial distension can be used as an early marker for the detection of arterial disease in murine models. The semi-automatic analysis method provided increased sensitivity to differences between experimental groups when compared to the manual analysis method.

Pre-stroke surgery is not beneficial to normotensive rats undergoing sixty minutes of transient focal cerebral ischemia.

Experimental stroke in rodents, via middle cerebral artery occlusion (MCAO), can be associated with a negative impact on wellbeing and mortality. In hypertensive rodents, pre-stroke craniotomy increased survival and decreased body weight loss post-MCAO. Here we determined the effect, in normotensive Sprague-Dawley rats following 60 minutes MCAO, with or without pre-surgical craniotomy, on post-stroke outcomes in terms of weight loss, neurological deficit, lesion volume and functional outcomes. There was no effect of pre-stroke craniotomy on indicators of wellbeing including survival rate (P = 0.32), body weight loss (P = 0.42) and neurological deficit (P = 0.75). We also assessed common outcome measures following experimental stroke and found no effect of pre-stroke craniotomy on lesion volume as measured by T2-weighted MRI (P = 0.846), or functional performance up to 28 days post-MCAO (staircase test, P = 0.32; adhesive sticker test, P = 0.49; cylinder test, P = 0.38). Thus, pre-stroke craniotomy did not improve animal welfare in terms of body weight loss and neurological deficit. However, it is important, given that a number of drug delivery studies utilise the craniotomy procedure, to note that there was no effect on lesion volume or functional outcome following experimental stroke.

Vitamin D3 supplementation of a high fat high sugar diet ameliorates prediabetic phenotype in female LDLR-/- and LDLR+/+ mice.

INTRODUCTION: Fatty liver disease is prevalent in populations with high caloric intake. Nutritherapeutic approaches are being considered, such as supplementary Vitamin D3 , to improve aspects of metabolic syndrome, namely fatty liver disease, hyperlipidemia, and insulin resistance associated with obesity. METHODS: We analyzed female LDLR-/- and LDLR+/+ mice on a 10-week diabetogenic diet for markers of fatty liver disease, metabolic strain, and inflammation. RESULTS: The groups on a high fat high sugar diet with supplementary Vitamin D3 , in comparison with the groups on a high fat high sugar diet alone, showed improved transaminase levels, significantly less hypertriglyceridemia and hyperinsulinemia, and histologically, there was less pericentral hepatic steatosis. Levels of non-esterified fatty acids and lipid peroxidation products were significantly lower in the group supplemented with additional Vitamin D3 , as were systemic markers of inflammation (serum endotoxin and IL-6). M2 macrophage phenotype predominated in the group supplemented with additional Vitamin D3 . Beneficial changes were observed as early as five weeks' supplementation with Vitamin D3 and extended to restoration of high fat high sugar diet induced decrease of bone mineral density. CONCLUSION: In summary, Vitamin D3 was a significantly beneficial dietary additive to blunt a prediabetic phenotype in diet-induced obesity of female LDLR-/- and LDLR+/+ mice.

An alternative surgical approach reduces variability following filament induction of experimental stroke in mice.

Animal models are essential for understanding the pathology of stroke and investigating potential treatments. However, in vivo stroke models are associated, particularly in mice, with high variability in lesion volume. We investigated whether a surgical refinement where reperfusion is not reliant on the Circle of Willis reduced outcome variability. Mice underwent 60 min of transient middle cerebral artery occlusion avoiding ligation of the external carotid artery. During reperfusion, the common carotid artery was either ligated (standard approach), or it was repaired to allow re-establishment of blood flow through the common carotid artery. All mice underwent MRI scanning for assessment of infarct volume, apparent diffusion coefficient and fractional anisotropy, along with terminal assessment of infarct volume by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Repairing the common carotid artery following middle cerebral artery occlusion enhanced reperfusion (P<0.01) and reduced the variability seen in both total (histological analysis, P=0.008; T2-weighted MRI, P=0.015) and core (diffusion tensor MRI, P=0.043) lesion volume. Avoiding external carotid artery ligation may improve animal wellbeing, through reduced weight loss, while using an alternative surgical approach that enabled reperfusion through the common carotid artery decreased the variability in lesion volume seen within groups.

Size distribution of air bubbles entering the brain during cardiac surgery.

BACKGROUND: Thousands of air bubbles enter the cerebral circulation during cardiac surgery, but whether high numbers of bubbles explain post-operative cognitive decline is currently controversial. This study estimates the size distribution of air bubbles and volume of air entering the cerebral arteries intra-operatively based on analysis of transcranial Doppler ultrasound data. METHODS: Transcranial Doppler ultrasound recordings from ten patients undergoing heart surgery were analysed for the presence of embolic signals. The backscattered intensity of each embolic signal was modelled based on ultrasound scattering theory to provide an estimate of bubble diameter. The impact of showers of bubbles on cerebral blood-flow was then investigated using patient-specific Monte-Carlo simulations to model the accumulation and clearance of bubbles within a model vasculature. RESULTS: Analysis of Doppler ultrasound recordings revealed a minimum of 371 and maximum of 6476 bubbles entering the middle cerebral artery territories during surgery. This was estimated to correspond to a total volume of air ranging between 0.003 and 0.12 mL. Based on analysis of a total of 18667 embolic signals, the median diameter of bubbles entering the cerebral arteries was 33 µm (IQR: 18 to 69 µm). Although bubble diameters ranged from ~5 µm to 3.5 mm, the majority (85%) were less than 100 µm. Numerous small bubbles detected during cardiopulmonary bypass were estimated by Monte-Carlo simulation to be benign. However, during weaning from bypass, showers containing large macro-bubbles were observed, which were estimated to transiently affect up to 2.2% of arterioles. CONCLUSIONS: Detailed analysis of Doppler ultrasound data can be used to provide an estimate of bubble diameter, total volume of air, and the likely impact of embolic showers on cerebral blood flow. Although bubbles are alarmingly numerous during surgery, our simulations suggest that the majority of bubbles are too small to be harmful.